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The photophysical properties of the β-barrel superfolder green fluorescent protein (sfGFP) arise from the chromophore that forms post-translationally in the interior of the protein. Specifically, the protonation state of the side chain of tyrosine 66 in the chromophore, in addition to the network of hydrogen bonds between the chromophore and surrounding residues, is directly related to the electronic absorbance and emission properties of the protein. The pH dependence of the photophysical properties of this protein were modulated by the genetic, site-specific incorporation of 3-nitro-l-tyrosine (mNO 2 Y) at site 66 in sfGFP. The altered photophysical properties of this noncanonical amino acid (ncAA) sfGFP construct were assessed by absorbance and fluorescence spectroscopies. Notably, a comparison of the p K a of the 3-nitrophenol side chain of mNO 2 Y incorporated in the protein relative to the phenol side chain of the tyrosine at site 66 in the native chromophore as well as the p K a of the 3-nitrophenol side chain of the free ncAA were measured and are compared. A structural analysis of the ncAA containing sfGFP construct is presented to yield molecular insight into the origin of the altered absorbance and fluorescence properties of the protein., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)